The goal of this proposal is to create rapid, inexpensive and robust whole-cell assays that can be performed in high throughput mode to allow the discovery of novel antibacterial molecules with specific mechanisms of action. These novel antibacterial molecules can provide the starting point for the discovery of new classes of antibiotics and in addition can provide the opportunity to further explore the cell biology of bacteria. The proposed studies start with highly sensitized S. aureus strains with well-defined conditional (heat sensitive) mutations in essential genes. These conditional lethal strains fail to grow in non-permissive growth conditions, yet grow reasonably well under permissive growth conditions. Semi-permissive conditions can be identified in which the strains show partial growth inhibition. Under these conditions, preliminary data have shown that certain strains fail to grow when challenged with an antibiotic at a concentration that does not inhibit the growth of the wild type strain. Furthermore, strains with mutations in different genes show selective hypersensitivity to different antibiotic compounds. The explanation for this phenomenon, partially supported by preliminary data, is that the combination of the defect caused by the genetic conditional lethal mutation combined with the chemical inhibition in a related biochemical pathway, causes a synthetic lethal combination. This phenomenon can be converted into a whole-cell, high throughput assay with target or pathway selectivity. Additional information is needed to develop the high throughput screens, the appropriate secondary assays and provide proof-of-principle for the general method. The specific experiments aim to develop a high throughput screen for each of 3 cellular pathways-DNA replication, cell wall biosynthesis and protein secretion. The specific semi-permissive conditions appropriate for conditional mutant strains with defects in genes of these pathways will be identified. Piloting of the screening method in 96-well format will be accomplished using antibiotics with known mechanisms of action, as well as a collection of diversity compounds. Hits from the preliminary screening will undergo genetic and biochemical secondary assays to further characterize the types of hits that arise, and the types of secondary assays needed to effectively sort compounds of interest from others that arise in the screen. These experiments should provide the specific information necessary to support the use of highly sensitized S. aureus conditional mutant strains in high throughput screens to identify inhibitors targeting gram positive cell wall biosynthesis, DNA replication and protein secretion, as well as provide more general understanding of the effective use of this type of whole cell screening.